Western Blot Chemiluminescent Substrate Selection Guide

Choose an ECL reagent for western blot detection

Chemiluminescence is the western blot detection method of choice in most protein laboratories, as it provides the greatest sensitivity and convenience for detection with film or digital imaging equipment. Several varieties of luminol-based chemiluminescent substrates are available, and choosing among them for a particular experiment or available equipment can seem overwhelming.

Chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the equal volumes of the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction produces light that can be detected by film or sensitive camera.

Protein detection technical handbook

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Protein Detection Technical Handbook

Criteria for choosing chemiluminescent substrates

Chemiluminescent substrates differ in price, ease-of-use, sensitivity (i.e., lower limit of detection) and compatibility with imaging equipment. More sensitive substrates cost more, so it might be natural to assume that one should use the most sensitive substrate that can be afforded. However, this is not necessarily correct because ease-of-use is an equally important consideration.

Although the actual procedure for preparing substrate working solution and applying it to a blot is practically identical for all chemiluminescent substrates, each substrate has unique requirements for optimization. In general, it is more difficult to optimize western blot protocols with more sensitive substrates because even the tiniest flaws in blocking or non-specific binding are detectable. No researcher wants to spend more time than absolutely necessary optimizing antibody dilutions and exposure times to obtain a useful result.

Available chemiluminescent substrate reagents

We offer five types of Thermo Scientific Pierce ECL and Thermo Scientific SuperSignal chemiluminescent substrates for Western blot detection with horseradish peroxidase enzyme (HRP):

  • Pierce ECL Substrate
    • A less expensive direct alternative to the once-popular GE Healthcare ECL (commonly called "Amersham ECL")
    • Appropriate for applications in which very abundant proteins (i.e., actin, tubulin, etc) are being probed or for experiments that have already been optimized with the GE Healthcare product
  • Pierce ECL Plus Substrate
    • Less-expensive direct alternative to GE Healthcare ECL Plus Substrate
    • Offers greater sensitivity and requires less antibody than ECL substrates
  • SuperSignal West Pico PLUS Chemiluminescent Substrate
    • Provides excellent performance for the range of target protein amounts typical of routine experiments
    • Stable signal duration over the critical 6 hour time period with usable light output up to 24 hours
  • SuperSignal West Dura Extended Duration Substrate
    • Offers high sensitivity and long-duration signal that facilitate CCD-camera and other digital imaging analysis
    • 24-hour signal output allows researchers to make multiple exposures with imaging equipment
  • SuperSignal West Femto Maximum Sensitivity Substrate
    • One of the most sensitive substrates available for Western blotting of any kind. When properly optimized, the lower detection limit rivals radioactive detection methods using 32P
    • However, without proper optimization, it easy to overwhelm the system with protein and enzyme, resulting in high background and false negative results

Chemiluminescent HRP substrates comparison chart

Sensitivity and antibody dilutions have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.

Thermo Scientific Substrate Primary Benefits Sensitivity Sensitivity
Signal Duration (hours) Recommended
Antibody Dilution
Pierce ECL Same signal and lower price than other ECL substrates Low picogram 1 1 to 2 1°: 0.2–10 μg/mL
2°: 0.07–1 μg/mL
Pierce ECL Plus Same signal and lower price than competing ECL Plus substrates Low picogram to mid-femtogram 3 5 1°: 0.05–1 μg/mL
2°: 5–40 ng/mL
SuperSignal West Pico PLUS Better signal and lower price than competing ECL substrates Low picogram to high femtogram 3 6 to 24 1°: 0.2–1 μg/mL
2°: 10–50 ng/mL
SuperSignal West Dura Best for use with imaging equipment Mid-femtogram 4 24 1°: 0.02–1 μg/mL
2°: 4–20 ng/mL
SuperSignal West Femto Most sensitive substrate for HRP detection Low to mid-femtogram 5 6 to 8 1°: 10–200 ng/mL
2°: 2–10 ng/mL
† Sensitivity Rating: 1=lowest, 5=highest

Related chemiluminescent reagents and kits

In addition to the reagent products listed above, complete western blotting kits are available for particular applications using Pierce ECL and SuperSignal Substrates. These specialty western blot detection kits include the chemiluminescent substrate plus validated components such as blocking buffer and secondary antibodies.

Also available are kits for direct in-gel detection of HRP-conjugated probes by chemiluminescence. Although not as sensitive as a typical Western blot, the in-gel system allows results to be obtained in times similar to the Fast Western (Thermo Scientific), SNAP i.d. (Millipore), and iBlot (Invitrogen) detection systems when used with compatible polyacrylamide gels. There is also the added advantage of reduced waste from buffers and consumables, such as filter paper and membranes.

Other specialized kits include those specific for glycosylation, His-tagged proteins far-Western assays.

Specific formulations and kits of SuperSignal Chemiluminescent Substrates are available for nucleic acid techniques, including EMSA and Southern blotting. These products depend upon the use of biotin-labeled DNA or RNA targets and hybridization probes, which are then detected via HRP-conjugated streptavidin.

Other instruments capable of ECL signal documentation

The following instruments listed here are capable of documenting signal from luminol-based substrates, such as the Thermo Scientific SuperSignal Substrates.

Supplier Instrument
ProteinSimple FluorChem imaging systems and AlphaImager HP imaging system
Bio-Rad ChemiDoc, ChemiDoc MP, ChemiDoc XRS+ imaging systems
Azure Biosystems c300, c400, c500 and c600 imaging systems, Sapphire Biomolecular Imager
Amersham Amersham Imager 600 series imaging systems
Li-COR Odyssey Fc imaging system and c-DiGit Blot Scanner
Syngene G:BOX and GeneGnome imaging systems